MECHANISMS OF IDIOTYPE SUPPRESSION I. In ... - BioMedSearch
MECHANISMS
OF IDIOTYPE
SUPPRESSION
I. In V i t r o G e n e r a t i o n o f Idiotype-Specific S u p p r e s s o r T Cells b y A n t i - I d i o t y p e A n t i b o d i e s a n d Specific Antigen* By BYUNG S. KIM From the Department of Microbiology-Immunology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611
Essentially all anti-phosphorylcholine (PC) a antibodies produced by BALB/c mice bear an idiotype that is indistinguishable from that of TEPC-15 myeloma protein (T15) (1, 2). These mice, therefore, are virtually tolerant to stimulation by PC antigen after a neonatal injection of antibodies specific to the T 15 idiotype (T15id). We have previously identified suppressor cells specific for the anti-PC response in idiotypically suppressed mice (3). The cells responsible for active suppression are represented in both the T and non-T-cell populations (3, 4). Similar suppressor cells have been also found in other systems of idiotype suppression (5, 6), suggesting that such idiotypespecific suppressor cells may play an important role in maintaining suppression, perhaps after the initial depletion of cell populations bearing specific Ig markers (7). In this study, the. mechanisms of idiotype suppression have been investigated by examining the generation of specific suppressor cells in vitro. We report here that anti-T15id antibodies, together with specific antigen, generate idiotype-specific suppressor T cells in vitro. This result suggests that suppressor T cells found in idiotypitally suppressed mice may have been similarly generated. Materials and Methods 7- to 10-wk-old BALB/e mice were purchased from Cumberland View Farms, Clinton, Tenn. Antigens. A rough strain of Streptococcus pneumoniae, R36a was obtained from the American Type Culture Collection, Roekville, Md. A vaccine of R36a was prepared by treatment with 0.5% formalin in 0.15 M NaC1 (3) and the vaccine was used as a PC antigen. As a control antigen, 2,4-dinitrophenyl-lysyl-Ficoll (Pharmacia Fine Chemicals, Div. of Pharmacia, Inc., Piseataway, N. J.) (DNP-Lys-FieolI), prepared by the method of Sharon et al. (8), was used throughout the experiments in this study. Antisera. The TEPC-15 myeloma protein (T15) was purified by using a PC-conjugated Sepharose 4B column (Pharmaeia Fine Chemicals, Div. of Pharmaeia Inc.) as described previously (4, 9). Anti-Tl5 antibodies were prepared in the ascitic fluid of A/He mice by multiple injections ofT15 protein according to the method of Tung et al. (10). The ascites was clarified by centrifugation and the fluid was further absorbed using immunoabsorbent columns conjugated with PC-binding myeloma proteins possessing different idiotypes (i.e. MOPC-167 Mice.
* Supported by Research grant R01 AI 15446from the U. S. Public Health Service. 1 Abbreviations used in this paper: Anti-id, anti-idiotype; BSS, Hank's balanced salt solution; DNP-Lys-
Ficoll, 2,4-dinitrophenyl-lysyl-Ficoll;KLH, keyhole limpet hemocyanin; PC, Phosphorylcholine; PFC, Plaque-forming cells; SRBC, sheep erythrocytes;T15, TEPC-15 myeloma protein; T15id, Idiotype of TEPC- 15 myelomaprotein. J. ExP.MED.© The RockefellerUniversityPress • 0022-1007/79/06/1371/08$1.00 Volume 149 June 1979 1371-1378
1371
1372
IDIOTYPE-SPECIFIC SUPPRESSOR T CELLS
and McPC-603). The resulting anti-idiotype (anti-T15id) ascitie fluid was specific for only T 15id when examined by radioimmunoassay using 12BI-T15 Fab, 125I-M603 Fab, and I~I-M 167 Fab: 20/~1 of a 1:100 dilution of the ascitic fluid precipitated 45% of T 15, but only 3% of M603 and 1% of M167 (details will be published elsewhere). A / H e mice were injected similarly with a mixture of Freund's complete adjuvant and saline without T15 protein. The resulting ascitic fluid was used as a control for anti-T15id ascites. Rabbit anti-mouse Ig was purchased from Miles Laboratories Inc., Miles Research Products, Elkhart, Ind. The antiserum was further absorbed with thymocytes of BALB/c mice. Pretreatment of normal BALB/e spleen cells with the anti-Ig serum (1:80) plus complement resulted in 92% reduction of the PFC to PC after in vitro stimulation. Anti-Thy 1.2 serum, prepared in A K R mice against C3H thymoeytes as described by Reif and Allen (11) was supplied by Dr. Nicholas M. Ponzio in our department. This antiserum (1: 50) did not reduce the anti-PC response of spleen cells, although 46% of the cells were killed by the treatment with this antiserum and agarose-absorbed rabbit complement (12). The detailed procedure of the treatment was described previously (3). Enrichment of B and T Cells. Essentially, the method of Julius et al. (13) was applied to enrich splenic B- and T-cell populations. Briefly, 1-2 × 10a spleen cells were loaded aseptically on a 10-ml nylon wool column equilibrated with RPMI-1640 (Grand Island Biological Co., Grand Island, N. Y.) supplemented with 2% fetal calf serum. After an incubation at 37°C for 45 min, a nonadherent cell population was separated from the column by adding warm (37°C) medium, dropwise. An adherent cell population was collected by forcing cold medium through the column using a plunger. Approximately 80% of the applied cells were recovered from the column and 50-60% of the recovered cells were in the T-cell-enriched fraction. Spleen Cell Cultures. The Mishell-Dutton technique of immunization of mouse spleen cells (14) was used, with the exception that the culture medium was RPMI-1640 with 25 mM Hepes buffer (Grand Island Biological Co.). Individual cultures contained 1.2 × 107 spleen cells and were immunized with either 2.5 × 10e pneumococei or 1 ng of DNP-Lys-Ficoll in 25 ~1. For induction of idiotype suppression, 100/~1 of a 1:200 dilution of anti-T 15id aseites in the culture medium was added to individual cultures. The final volume of individual cultures was adjusted to 1 ml. Unless otherwise indicated, the cultures were incubated for 4 d to elicit immune responses in vitro. Generation of Suppressor Cells In Vitro. Normal BALB/c spleen cells (1.2 × 107) were cultured for 3 d in a multiwell tissue culture plate (Falcon 3008, Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard, Calif.) with a 1:2,000 final dilution of anti-T15id ascites and 2.5 × 106 R36a cells as described above. The precultured cells were washed three times with Hank's balanced salt solution (BSS) and then cocultured with 1.2 × 107 freshly prepared normal BALB/c spleen cells in the presence of the same quantity of R36a. After 4 d of additional incubation, immune responses of individual cultures to PC were determined by a hemolytic plaque assay. Hemolytic Plaque Assay. The number of plaque-forming cells was determined by a slide modification of the Jerne-Nordin hemolytic plaque technique (15). To detect plaque-forming cells (PFC) to PC, sheep erythroeytes (SRBC) were coated with R36a C-polysaccharide employing chromic chloride (16) and used as the specific target cells (1). For target cells of DNP-specific PFC, SRBC were conjugated with trinitrobenzene sulfonic acid to yield TNPSRBC (17). Enumeration of PFC Producing Anti-PC Antibodies with Tl5id. The number of PFC secreting antibodies bearing T15id was determined by the method of Cosenza and K6hler (18). Spleen ceils immunized with R36a for 4 d in vitro were incubated for 1 h with a 1:400 final dilution of anti-T15id ascites in BSS. The number of plaques was counted after an additional 1-h incubation with complement, and compared with the number of plaques developed in the absence of anti-T 15id antibody. Results
In Vitro Generation of Idiotype-Specific Suppressor Cells. T h e possibility t h a t antii d i o t y p e (anti-id) a n t i b o d i e s were involved in the generation o f idiotype-speeific
BYUNG S. KIM
1373
TABLE I
Generation of Idiotype-Specific Suppressor Cells In Vitro by Treatment with Anti-ldiotype Antibodies PFC/culture
Spleen cells/culture* Precultured
Fresh normal
PC total*
0 0 1.2 x 107 + control 1.2 x 107 + anti-Tl5id
2.4 X 10 7 1.2 x 107 1.2 x 107 1.2 x 10T
3,520 + 130 2,510 :l: 270 1,130:1:130 65 -4- 35
TI5(+)§ % 97 98 93 1
DNP t o t a l 2,016:1:207 2,620:1:331 1,423 :t: 338 1,945 :t: 261
* Normal BALB/c spleen cells were incubated for 3 d with either control or anti-Tl5id ascitic fluid (1: 2,000) in the presence of an immunizing dose (2.5 × 10e cells) of R36a. The treated cells were washed three times with BSS and then added to fresh normal BALB/c spleen cells. Individual cultures were adjusted t o a 1-ml final volume. The cell mixture was incubated for 4 d with either 2.5 X 106 R36a or 1 ng of DNP-Lys-Ficoll. :~Individual cultures were assayed for their specific PFC using either PC-SRBC or TNP-SRBC, respectively. The number of PFC per culture represents the geometric mean and standard error of determinations for individual triplicate cultures. § The number of PFC producing anti-PC antibodies with Tl5id was determined by specific inhibition of plaque formation by treatment with anti-T15id antibodies. suppressors was examined by direct treatment of normal spleen cells in vitro with anti-id antibodies. T o determine the suppressor cell activity, normal spleen cells that were incubated for 3 d with anti-T15id antibodies and R 3 6 a were subsequently washed and cultured with fresh normal B A L B / c spleen cells in the presence of R 3 6 a or a control antigen, DNP-Lys-Ficoll. T h e spleen cells which had been preincubated with anti-T15id antibodies were capable o f suppressing the anti-PC response o f fresh normal spleen cells, whereas spleen cells treated with the control ascites were not inhibitory (Table I). T h e suppression was specific to PC because those precultured cells did not inhibit the i m m u n e response of normal spleen cells to DNP-Lys-Ficoll. These results indicated that preincubation o f normal spleen cells with anti-T15id antibodies resulted in generation o f a suppressor cell population that was specific with the anti-PC response. Idiotype analysis was performed using the plaque-inhibition technique (18) to examine the specificity o f idiotype suppression mediated by the in vitro generated suppressor cells (Table I). Either spleen cell cultures of normal B A L B / c mice or those receiving spleen cells pretreated with R36a and control ascites, induced a normal level of P F C producing anti-PC antibodies with T l 5 i d : the n u m b e r of T15id-producing PFC was >93% of the total PFC. In contrast, B A L B / c spleen cells cocultured with suppressor cells were able to m o u n t neither a normal level of anti-PC response (
OF IDIOTYPE
SUPPRESSION
I. In V i t r o G e n e r a t i o n o f Idiotype-Specific S u p p r e s s o r T Cells b y A n t i - I d i o t y p e A n t i b o d i e s a n d Specific Antigen* By BYUNG S. KIM From the Department of Microbiology-Immunology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611
Essentially all anti-phosphorylcholine (PC) a antibodies produced by BALB/c mice bear an idiotype that is indistinguishable from that of TEPC-15 myeloma protein (T15) (1, 2). These mice, therefore, are virtually tolerant to stimulation by PC antigen after a neonatal injection of antibodies specific to the T 15 idiotype (T15id). We have previously identified suppressor cells specific for the anti-PC response in idiotypically suppressed mice (3). The cells responsible for active suppression are represented in both the T and non-T-cell populations (3, 4). Similar suppressor cells have been also found in other systems of idiotype suppression (5, 6), suggesting that such idiotypespecific suppressor cells may play an important role in maintaining suppression, perhaps after the initial depletion of cell populations bearing specific Ig markers (7). In this study, the. mechanisms of idiotype suppression have been investigated by examining the generation of specific suppressor cells in vitro. We report here that anti-T15id antibodies, together with specific antigen, generate idiotype-specific suppressor T cells in vitro. This result suggests that suppressor T cells found in idiotypitally suppressed mice may have been similarly generated. Materials and Methods 7- to 10-wk-old BALB/e mice were purchased from Cumberland View Farms, Clinton, Tenn. Antigens. A rough strain of Streptococcus pneumoniae, R36a was obtained from the American Type Culture Collection, Roekville, Md. A vaccine of R36a was prepared by treatment with 0.5% formalin in 0.15 M NaC1 (3) and the vaccine was used as a PC antigen. As a control antigen, 2,4-dinitrophenyl-lysyl-Ficoll (Pharmacia Fine Chemicals, Div. of Pharmacia, Inc., Piseataway, N. J.) (DNP-Lys-FieolI), prepared by the method of Sharon et al. (8), was used throughout the experiments in this study. Antisera. The TEPC-15 myeloma protein (T15) was purified by using a PC-conjugated Sepharose 4B column (Pharmaeia Fine Chemicals, Div. of Pharmaeia Inc.) as described previously (4, 9). Anti-Tl5 antibodies were prepared in the ascitic fluid of A/He mice by multiple injections ofT15 protein according to the method of Tung et al. (10). The ascites was clarified by centrifugation and the fluid was further absorbed using immunoabsorbent columns conjugated with PC-binding myeloma proteins possessing different idiotypes (i.e. MOPC-167 Mice.
* Supported by Research grant R01 AI 15446from the U. S. Public Health Service. 1 Abbreviations used in this paper: Anti-id, anti-idiotype; BSS, Hank's balanced salt solution; DNP-Lys-
Ficoll, 2,4-dinitrophenyl-lysyl-Ficoll;KLH, keyhole limpet hemocyanin; PC, Phosphorylcholine; PFC, Plaque-forming cells; SRBC, sheep erythrocytes;T15, TEPC-15 myeloma protein; T15id, Idiotype of TEPC- 15 myelomaprotein. J. ExP.MED.© The RockefellerUniversityPress • 0022-1007/79/06/1371/08$1.00 Volume 149 June 1979 1371-1378
1371
1372
IDIOTYPE-SPECIFIC SUPPRESSOR T CELLS
and McPC-603). The resulting anti-idiotype (anti-T15id) ascitie fluid was specific for only T 15id when examined by radioimmunoassay using 12BI-T15 Fab, 125I-M603 Fab, and I~I-M 167 Fab: 20/~1 of a 1:100 dilution of the ascitic fluid precipitated 45% of T 15, but only 3% of M603 and 1% of M167 (details will be published elsewhere). A / H e mice were injected similarly with a mixture of Freund's complete adjuvant and saline without T15 protein. The resulting ascitic fluid was used as a control for anti-T15id ascites. Rabbit anti-mouse Ig was purchased from Miles Laboratories Inc., Miles Research Products, Elkhart, Ind. The antiserum was further absorbed with thymocytes of BALB/c mice. Pretreatment of normal BALB/e spleen cells with the anti-Ig serum (1:80) plus complement resulted in 92% reduction of the PFC to PC after in vitro stimulation. Anti-Thy 1.2 serum, prepared in A K R mice against C3H thymoeytes as described by Reif and Allen (11) was supplied by Dr. Nicholas M. Ponzio in our department. This antiserum (1: 50) did not reduce the anti-PC response of spleen cells, although 46% of the cells were killed by the treatment with this antiserum and agarose-absorbed rabbit complement (12). The detailed procedure of the treatment was described previously (3). Enrichment of B and T Cells. Essentially, the method of Julius et al. (13) was applied to enrich splenic B- and T-cell populations. Briefly, 1-2 × 10a spleen cells were loaded aseptically on a 10-ml nylon wool column equilibrated with RPMI-1640 (Grand Island Biological Co., Grand Island, N. Y.) supplemented with 2% fetal calf serum. After an incubation at 37°C for 45 min, a nonadherent cell population was separated from the column by adding warm (37°C) medium, dropwise. An adherent cell population was collected by forcing cold medium through the column using a plunger. Approximately 80% of the applied cells were recovered from the column and 50-60% of the recovered cells were in the T-cell-enriched fraction. Spleen Cell Cultures. The Mishell-Dutton technique of immunization of mouse spleen cells (14) was used, with the exception that the culture medium was RPMI-1640 with 25 mM Hepes buffer (Grand Island Biological Co.). Individual cultures contained 1.2 × 107 spleen cells and were immunized with either 2.5 × 10e pneumococei or 1 ng of DNP-Lys-Ficoll in 25 ~1. For induction of idiotype suppression, 100/~1 of a 1:200 dilution of anti-T 15id aseites in the culture medium was added to individual cultures. The final volume of individual cultures was adjusted to 1 ml. Unless otherwise indicated, the cultures were incubated for 4 d to elicit immune responses in vitro. Generation of Suppressor Cells In Vitro. Normal BALB/c spleen cells (1.2 × 107) were cultured for 3 d in a multiwell tissue culture plate (Falcon 3008, Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard, Calif.) with a 1:2,000 final dilution of anti-T15id ascites and 2.5 × 106 R36a cells as described above. The precultured cells were washed three times with Hank's balanced salt solution (BSS) and then cocultured with 1.2 × 107 freshly prepared normal BALB/c spleen cells in the presence of the same quantity of R36a. After 4 d of additional incubation, immune responses of individual cultures to PC were determined by a hemolytic plaque assay. Hemolytic Plaque Assay. The number of plaque-forming cells was determined by a slide modification of the Jerne-Nordin hemolytic plaque technique (15). To detect plaque-forming cells (PFC) to PC, sheep erythroeytes (SRBC) were coated with R36a C-polysaccharide employing chromic chloride (16) and used as the specific target cells (1). For target cells of DNP-specific PFC, SRBC were conjugated with trinitrobenzene sulfonic acid to yield TNPSRBC (17). Enumeration of PFC Producing Anti-PC Antibodies with Tl5id. The number of PFC secreting antibodies bearing T15id was determined by the method of Cosenza and K6hler (18). Spleen ceils immunized with R36a for 4 d in vitro were incubated for 1 h with a 1:400 final dilution of anti-T15id ascites in BSS. The number of plaques was counted after an additional 1-h incubation with complement, and compared with the number of plaques developed in the absence of anti-T 15id antibody. Results
In Vitro Generation of Idiotype-Specific Suppressor Cells. T h e possibility t h a t antii d i o t y p e (anti-id) a n t i b o d i e s were involved in the generation o f idiotype-speeific
BYUNG S. KIM
1373
TABLE I
Generation of Idiotype-Specific Suppressor Cells In Vitro by Treatment with Anti-ldiotype Antibodies PFC/culture
Spleen cells/culture* Precultured
Fresh normal
PC total*
0 0 1.2 x 107 + control 1.2 x 107 + anti-Tl5id
2.4 X 10 7 1.2 x 107 1.2 x 107 1.2 x 10T
3,520 + 130 2,510 :l: 270 1,130:1:130 65 -4- 35
TI5(+)§ % 97 98 93 1
DNP t o t a l 2,016:1:207 2,620:1:331 1,423 :t: 338 1,945 :t: 261
* Normal BALB/c spleen cells were incubated for 3 d with either control or anti-Tl5id ascitic fluid (1: 2,000) in the presence of an immunizing dose (2.5 × 10e cells) of R36a. The treated cells were washed three times with BSS and then added to fresh normal BALB/c spleen cells. Individual cultures were adjusted t o a 1-ml final volume. The cell mixture was incubated for 4 d with either 2.5 X 106 R36a or 1 ng of DNP-Lys-Ficoll. :~Individual cultures were assayed for their specific PFC using either PC-SRBC or TNP-SRBC, respectively. The number of PFC per culture represents the geometric mean and standard error of determinations for individual triplicate cultures. § The number of PFC producing anti-PC antibodies with Tl5id was determined by specific inhibition of plaque formation by treatment with anti-T15id antibodies. suppressors was examined by direct treatment of normal spleen cells in vitro with anti-id antibodies. T o determine the suppressor cell activity, normal spleen cells that were incubated for 3 d with anti-T15id antibodies and R 3 6 a were subsequently washed and cultured with fresh normal B A L B / c spleen cells in the presence of R 3 6 a or a control antigen, DNP-Lys-Ficoll. T h e spleen cells which had been preincubated with anti-T15id antibodies were capable o f suppressing the anti-PC response o f fresh normal spleen cells, whereas spleen cells treated with the control ascites were not inhibitory (Table I). T h e suppression was specific to PC because those precultured cells did not inhibit the i m m u n e response of normal spleen cells to DNP-Lys-Ficoll. These results indicated that preincubation o f normal spleen cells with anti-T15id antibodies resulted in generation o f a suppressor cell population that was specific with the anti-PC response. Idiotype analysis was performed using the plaque-inhibition technique (18) to examine the specificity o f idiotype suppression mediated by the in vitro generated suppressor cells (Table I). Either spleen cell cultures of normal B A L B / c mice or those receiving spleen cells pretreated with R36a and control ascites, induced a normal level of P F C producing anti-PC antibodies with T l 5 i d : the n u m b e r of T15id-producing PFC was >93% of the total PFC. In contrast, B A L B / c spleen cells cocultured with suppressor cells were able to m o u n t neither a normal level of anti-PC response (
